GIRARDEAU Paul SILVESTRE DE FERRON Benoit. Synaptic transmission is limited by the recycling of synaptic vesicles for many rounds of use Synaptic transmission

A READILY RETRIEVABLE POOL OF SYNAPTIC VESICLES

GIRARDEAU Paul

SILVESTRE DE FERRON Benoit

Synaptic transmission is limited by the recycling of synaptic vesicles for many rounds of use

Introduction Synaptic transmission

The main process of vesicle recycling is mediated by clathrin

The formation of clathrin-coated vesicles from the plasma membrane

BUT slow process (sec to mn)

Introduction Synaptic transmission

2 mechanisms identified

Continuous activity Necessity of rapid recycling

Clathrin mediated : Preassembled structures

Pool of preassembled vesicle proteins

At the pre-synaptic surface

Introduction Vesicle recycling

Clathrin-independent : Kiss and run

What are the temporal dynamics of such a « readily retrievable pool » ?

VARIOUS SYNAPTIC PROTEINS AT THE SYNAPTIC VESICULAR MEMBRANE

Synatptotagmin (Syt1) and Synaptobrevin (Syb2) : most numerous vesicular proteins VGAT (vesicular transporter of amino acids in inhibitory neurons)

Labeling:• of Syt1, Syb 2 and VGAT• to test the hypothesis of the pool of preassembled structures • in rat hippocampal neurons

Introduction Vesicle proteins

Methods Exo-endoxytosis of endogenous synaptic vesicle proteins

Synaptotagmin 1 antibody coupled with cypHer (pH sensitive fluorophore) anti-Syt1-cypHer

VGAT antibody coupled with cypHer (pH sensitive fluorophore)

anti-VGAT-cypHer

Normalized fluorescence of anti-Syt1-cypHer

The fluorescence is maximal at pH 5.5(pH of inside synaptic vesicle)

Cypher emits red fluorescence when excited at 640 nm

For studying inhibitory synapses

7,47,37,27,17,06,96,86,76,66,56,46,36,26,16,05,95,85,75,6

5,5

pH

Synaptotagmin 1 antibody coupled with cypHer (pH sensitive fluorophore)

Synaptotagmin 1 Red fluorescence emission

Spontaneous activity

Results Exo-endoxytosis of endogenous synaptic vesicle proteins

5.5

pH

VGAT antibody coupled with cypHer (pH sensitive fluorophore)

VGAT Red fluorescence emission


Fluorescence image of hippocampal neurons labeled with anti-VGAT-cypHer


Question Exo-endoxytosis of endogenous synaptic vesicle proteins

Are these probes efficiency to report stimulation-dependent exo-endocytosis?

7,4

7,37,27,17,06,96,86,76,66,56,46,36,26,16,05,95,85,75,6

5,5

pH

Synaptotagmin 1 antibody coupled with cypHer (pH sensitive fluorophore)-> Anti-Syt1-cypHer

Synaptotagmin 1

Red fluorescence emission

Elicited APs at 20 Hz(50, 200, 600, 900)


7,4

7,37,27,17,06,96,86,76,66,56,46,36,26,16,05,95,85,75,6

5,5

pH


Synaptotagmin 1



Elicited APs at 20 Hz(50, 200, 600, 900)

Results

These probes are efficiency to report stimulation dependent exo-endocytosis

Exo-endoxytosis of endogenous synaptic vesicle proteins

50 APs 100 APs 200 APs

Results Size of the surface pool of synaptic vesicle constituents

200 APs

Previous studies :Endocytic rate after stimulus = Endocytic rate during the stimulus

Fluorescence variations rise linearly with stimulus strength

Question Size of the surface pool of synaptic vesicle constituents

Are endogenous synaptic vesicle proteins already present on the presynaptic membrane?

7,4

7,37,27,17,06,96,86,76,66,56,46,36,26,16,05,95,85,75,6

5,5

pH


Synaptotagmin 1


Buffer of pH 5,5

Buffer of pH 5,5Proteins already presents

on the presynaptic membrane

Num

ber o

f bou

tons

Results Size of the surface pool of synaptic vesicle constituents

These proteins are able of compensating exocytosis induced by 70 APs

ΔF ~ 50 a.u.

ΔF = size of the surface poolΔF

Methods Labeled antibodies report same recycling kinectics as spH

Dual-color imaging

Exogenous probe

Overexpressed probe

Synaptotagmin 1 antibody coupled with cypHer (pH sensitive fluorophore) anti-Syt1-cypHer

Fluorescence in acidic compartments

Synaptobrevin 2 coupled with pHluorin (pH sensitive GFP) SpH

Fluorescence in neutral compartments

200 APs at 20 Hz

Synaptotagmin 1



Synaptobrevin 2 coupled with pHluorin (pH sensitive fluorophore)-> SpH

Green fluorescence emission

pH

5,5

7,4

Results Labeled antibodies report same recycling kinectics as spH

Synaptotagmin 1



pH

5,5

7,4


Green fluorescence emission

Mirror-image signals

200 APs at 20 Hz


These probes report exo-endocytosis


Same results in inhibitory synapses with VGAT

Question A surface RRetP of synaptic vesicle constituents

Are synaptic vesicle proteins, exo- and endocytosed by the same stimulus,

identical or different?

Vesicle proteins already present on the presynaptic membrane

Methods A surface RRetP of synaptic vesicle constituents

Inactivation of Synaptobrevin 2 fluorescence on the presynaptic membrane TEV cleavage site between Synaptobrevin 2 and pHluorin

Inactivation of Synaptotagmin 1 fluorescence on the vesicles Photobleaching

First experiment

Second experiment

Synaptotagmin 1


pH

5,5

7,4


TEV cleavage site

pH

Tobacco etch virus (TEV) protease

50 APs at 20 Hz

A surface pool of vesicle proteins is

endocytosed

Results A surface RRetP of synaptic vesicle constituents

Synaptotagmin 1


pH

5,5

7,4


pH

Photobleaching

50 APs at 20 Hz

Presorted synaptic vesicle proteins are preferentially endocytosed on exocytosis



Endocytosis of Syt1 or VGAT is not perturbed by spH overexression

Without spH overexpression

With spH overexpression


Readily Retrievable Pool (RRetP)

RRP seems to be counterbalanced by an RRetP

of similar size

Readily Releasable Pool (RRP)

Partial bleaching of the surface pool

Question Spatial organization of the RRetP

How is the functionnal surface pool spatially organized at the presynapse?

Methods Spatial organization of the RRetP

Antibody to Synaptotagmin 1

Secondary antibody

Antibody to Synaptotagmin 1

Secondary antibody

Antibody to RIM (Active Zone)

Secondary antibody

Antibody to Homer1 (Post Synaptic Density)

Secondary antibody

First labeling Second labeling

IsoSTED microscopy4Pi microscopy

Results Spatial organization of the RRetP

H

H

H

R

R

R

Presynaptic PostsynapticR

H

RIM (AZ)

Homer1 (PSD)

Syt1 (?)

?

IsoSTED

Results Spatial organization of the RRetP

H

H

H

R

R

R

?Presynaptic Postsynaptic

R

H

RIM

Homer1

Syt1Axon

d

α

Doghnut-like arrangement around the active zone of the RRetP

Discussion and conclusion

Vesicle proteins already presents on the presynaptic membrane

Presorted synaptic vesicle proteins are preferentially endocytosed on exocytosis

Doghnut-like arrangement around the active zone of the RRetP

RRP seems to be counterbalanced by an RRetP of similar size

Discussion and conclusion


200 APs

50 APs

Incomplete depletion of the RRetP during the 1st stimulus

Partial replenishment of the RRetP after the 1st stimulus

Documents

GIRARDEAU Paul SILVESTRE DE FERRON Benoit. Synaptic transmission is limited by the recycling of synaptic vesicles for many rounds of use Synaptic transmission